The present invention relates to carbonate ionophores and carbonate sensors having membranes containing active amounts of these ionophores.
Carbon dioxide, the major end product of the metabolism of foodstuffs, is the most commonly measured initial parameter in evaluating the body""s ability to control pH. Carbonate (CO3=) and bicarbonate (HCO3xe2x88x92) anions in blood arise from dissolved carbon dioxide (CO2). The relative amounts CO3=, HCO3xe2x88x92 and CO2 are determined by equilibrium constants controlling the dissolution and dissociation of CO2. This equilibrium system buffers blood, that is, it limits changes in blood pH that could occur upon entry of acid or base. Physiology controls the excretion of CO2 through changes in the rate and depth of respiration. Knowledge of the CO3=, and therefore CO2, concentration in the blood permits an approximation of the acid-base balance and aids in elucidating abnormal conditions. The measurement of the carbonate ion concentration in a blood, serum or plasma sample is a crucial test for clinical diagnosis.
Electrochemical sensor technology is an advantageous approach to measure the carbonate ion concentration in patient samples. Such technology is not subject to interferences that affect optically based technologies. A subset of electrochemistry is potentiometry, wherein the potential is measured at an ion-selective membrane interposed between a sample and a reference solution. Typically the ion-selective membrane comprises a polymer matrix, a plasticizer and an ionophore. The membrane so prepared captures the ions of interest in an amount proportional to the concentration of ion in the sample, produces a corresponding potential change and is assembled with appropriate electrical connections and reference solution to construct an ion-selective electrode (ISE). This ISE, together with a sample-contacting reference electrode, forms an electrochemical cell capable of measuring potentials that may be related to ion concentration in the sample.
A further development in ion-selective electrode technology is the solid-state sensor. A solid-state ISE does not utilize a solution-phase reference, reducing the size and complexity of the device. In addition, the design facilitates mass fabrication. Solid state sensor technology has been described in the patent literature, see for instance U.S. Pat. Nos. 5,522,978; 5,284,568; and 4,933,048.
Carbonate ionophores and carbonate sensor technology have been described in the scientific and patent literature. Early examples include Analytical Chemistry 41 (1969) 1128 and U.S. Pat. No. 3,723,281. Both of these possessed relatively low selectivity over chloride anion. Another example, Analytica Chimica Acta 76 (1975) 155, reported data demonstrating better selectivity over chloride, sulfate and phosphate, but utilized a liquid membrane, not a physically stable polymeric membrane. Meyerhoff and Greenberg report a carbonate-selective polymer membrane electrode in Analytica Chimica Acta 141 (1982) 57, but this sensor is subject to interference from salicylate anion, a metabolite of aspirin. Overcoming interference from salicylate is important for clinical applications.
U.S. Pat. No. 4,810,351 describes an ISE with improved selectivity for carbonate over salicylate, but the ISE is of the traditional configuration and requires an internal reference solution. U.S. Pat. No. 5,174,872 and Clinical Chemistry 32 (1986) 137 describe the addition of salicylate-complexing agents to samples in order to improve the apparent selectivity of carbonate ISEs. Additional approaches to overcoming salicylate interference include those described in Analytical Chemistry 65 (1993) 3151; U.S. Pat. No. 4,272,328; US statutory invention registration H745, and WO 9838503. Each of these constitutes a multilayered membrane structure or multilayered membrane with an internal filling solution, rendering the device more complex and less amenable to mass fabrication processes.
One of the major technical obstacles against commercialization of carbonate sensors is the lack of highly selective carbonate ionophores to overcome the interference of salicylate ion, a metabolite of commonly used drug aspirin. The present invention comprises a novel ionophore with improved selectivity of carbonate ions over salicylate ions. The major difference between the new ionophore and the previously known carbonate ionophores, such as described in Analytica Chimica Acta 233 (1990) 41, is the introduction of the amide group at the ortho-position of the benzene ring adjacent to the active site of the ionophore (trifluoroacetyl group). The amide group makes the trifluoroacetyl group (the active site for carbonate ion) not only more reactive, but also more sterically hindered. Enhancement of steric hindrance is thought to be desirable because the active site will be more selective to a smaller molecule, like a carbonate ion, than a larger molecule, such as salicylate ion. Such a selectivity improvement does not entail complex, multilayer membrane structures nor extensive sample pretreatment with additives. The disclosed ionophore is believed to be a novel and useful composition of matter.
It is an objective of this invention to provide novel ionophores for carbonate-selective electrodes possessing improved selectivity over salicylate, and other interfering anions. It is a further objective to describe the preparation of polymeric membranes containing the novel ionophores. Novel carbonate ionophores were synthesized and used in a solid-state sensor membrane to detect carbonate concentration in biological samples. The sensor membrane containing this novel ionophore demonstrated surprising selectivity for carbonate over other ions, especially for salicylate ion. Salicylate interference in clinical samples has been a major obstacle for developing a successful carbonate sensor. In addition, the sensor prepared with this ionophore has shown good use life, retaining at least 80% sensitivity.
The invention includes novel ionophores of the formula: 
where R1, R2, R3 and R4 are independent straight or branched chain alkyl groups having 4 to 12 carbon atoms or the alkyl groups may optionally contain a cycloalkyl group having 3 to 8 carbon atoms or R1 and R2 or R3 and R4 together with N to which they are attached, can form a heterocycle ring having 5 to 8 carbon atoms.
In one embodiment, the compound depicted above is substituted at R1, R2, R3, and R4 with straight or branched chain alkyl groups having 4 to 12 carbon atoms.
In another embodiment, the compound depicted above is substituted at R1, R2, R3 and R4 with straight or branched chain alkyl groups having 6 to 10 carbon atoms.
In a most preferred embodiment, the compound depicted above is substituted at R1, R2, R3, and R4 with n-octyl groups.
Thus, the present invention includes sensors having a membrane with an ionophore for detecting carbonate ion in a test sample, the improvement comprising the addition of an effective amount of the above-described compound as the ionophore. Preferably, the ionophore for detecting carbonate is an effective amount of the compound depicted above as Formula 1 substituted at R1, R2, R3, and R4 with n-octyl groups. The term effective amount is intended to define the amount of ionophore required to provide good use life, retaining at least 80% sensitivity over a period from about two-weeks to about two months long.